When analysing a biological or chemical sample, the detection of glycosidase activity may be most useful (Boonacker E. and Van Noorden C. J. F. (2001) J. Histochem. Cytochem. 49, 1473-1486; Perry, J. D., James, A. L., Morris, K. A., Oliver, M., Chilvers, K. F., Reed, R. H., & Gould, F. K. (2006). Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci. Journal of Applied Microbiology, 101(5), 977-985; Orenga, S., James, A. L., Manafi, M., Perry, J. D., & Pincus, D. H. (2009). Enzymatic substrates in microbiology. Journal of Microbiological Methods, 79(2), 139-155). Whole organisms, cells or cell extracts, biological liquids or chemical mixtures are examples of biological or chemical samples in which glycosidase activity can be detected. Glycosidases form a vast family of enzymes including numerous biomarkers of various pathologies. They are also involved in numerous benign cell processes and are the subject of countless research work by cell biologists. The detection thereof may therefore afford information on a particular metabolic or morbid condition for example.
On this account, a probe capable of detecting glycosidase activity would be most useful. The detection of this activity by capturing fluorescence light is a much more sensitive technique than collecting remaining white light after mere absorption by a probe i.e. the detection threshold is much lower. The detection of fluorescence emission is very easy to carry out which means that fluorescent probes are tools of great interest for life sciences. For example, the class of fluorophores leading to Excited State Intramolecular Proton Transfer (ESIPT) is described in particular in a) Ormson, S. M., et al. Progress in Reaction Kinetics (1994) 19, 45-91; b) Legourrierec, D., et al. Progress in Reaction Kinetics (1994), 19, 211-275; et c) Zhao, J., Ji, S., Chen, Y., Guo, H., & Yang, P. (2012). Excited state intramolecular proton transfer (ESIPT): from principal photophysics to the development of new chromophores and applications in fluorescent molecular probes and luminescent materials. Physical chemistry chemical physics, 14(25), 8803). The first interpretation of high fluorescence found in some phenolic compounds as being an ESIPT phenomenon can be attributed to Weller (for methyl salicylate: Weller, A. (1961). Fast Reactions of Excited Molecules. Progress in Reaction Kinetics and Mechanism 1, 187), and to Heller and Williams (for hydroxyphenylbenzoxazoles: Heller A., and Williams, D. L., J. Phys. Chem. (1970)74, 4473-4480).
The ESIPT class of fluorophores is particularly appealing for researchers in life sciences on account of its exceptional properties compared with conventional fluorophores. The exceptional properties of ESIPT fluorophores are:
(a) large Stokes shift often exceeding 130 nm and capable of reaching values of 250 nm, thereby allowing choices of instruments maximizing detection sensitivity;
(b) excellent resistance to photobleaching with rates possibly having higher orders of magnitude than those of standard fluorophores such as fluorescein;
(c) the possibility of designing fluorophores which emit brilliant fluorescence in the solid state, a rare property among all known fluorophores. This latter capability allows a signal of high intensity to be produced at the probe activation site, with minimal dilution due to scattering; and finally
(d) the possibility of designing ESIPT fluorophores which emit in the red or near infrared (600 to 850 nm) where tissue transparency is highest; the corresponding probe would then be particularly suitable for imaging in living animals.
Most of these properties, if such fluorophore were to be integrated in a probe, would make a major contribution compared with the properties of conventional commercially available probes the action of which is hindered by small Stokes shifts and medium to high photobleaching, inevitably leading to fluorophores in solution state.
In the invention, the inventor focused on the judicious choice of a repeat unit cleavable by a glycosidase enzyme which would impart the complete probe, incorporating an ESIPT fluorophore as described above, with total water-solubility a prerequisite in order to reach the sites and tissues of interest. Said probe would allow a significant increase in detection sensitivity, which on this account would allow a reduction in dose and thereby would be particularly suitable for application to in vivo imaging whilst reducing toxicity problems. The level of sensitivity is closely related to: (i) photobleaching rate, (ii) the extent of accumulation of the fluorescent signal at its production site (and hence the rate of scattering from this site, and the issue of knowing whether or not the fluorophore precipitates,) (iii) the true off/on mode for probe functioning (no background noise due to non-converted fluorescence of the probe and (iv) the extent of superimposition of the excitation spectrum and emission spectrum (their separation at the baseline being the most favourable configuration; see point (a) above). Point (iv) is of particular importance since complete separation at the baseline provides the opportunity of selecting very wide filters for the light source (to excite the molecule at every possible wavelength), but more importantly for the detector (to collect photons derived from all the wavelengths emitted by the fluorophore). Point (iv) also minimises perturbation of the detection process by tissue autofluorescence (characterized by the small Stokes shift of natural fluorophores), a recurrent problem encountered with known fluorophores which also have a small Stokes shift.
In the recent years there has been increasing interest in the development of three-component initiator/spacer/fluorophore enzyme substrates, using self-immolative spacers as linkers. The important class of ESIPT fluorophores includes dichloro-HPQ (6-chloro-2-(5-chloro-2-hydroxyphenyl)-4(3H)-Quinazolinone; CAS number: 28683-92-3) that is of particular interest since it is fully insoluble in aqueous/physiological media whilst being highly fluorescent in the solid state and only in the solid state. Nonetheless, it is very difficult to use dichloro-HPQ when developing a molecular probe to provide data on the activity of a glycosidase. In addition, the chief activities for which an HPQ-based probe has already been developed (and marketed) are those of phosphatases, due to the fact that it is impossible to create stable HPQ-based probes with a glycosylated phenolic hydroxyl since the resulting product has a propensity for rapid spontaneous hydrolysis which evidently releases free insoluble dichloro-HPQ thereby producing an erroneous signal (“background signal”). It is also to be noted that the marketing by Molecular Probes of such glycosylated compounds (ELF 97 glucuronidase substrate (No. E6587) and ELF 97 chitinase/N-acetylglucosaminidase substrate (No. E22011) was interrupted in 2008 and the study thereof by some researchers has not been continued (a) Chen, K.-C., Wu, C.-H., Chang, C.-Y., Lu, W.-C., Tseng, Q., Prijovich, Z. M., Schechinger, W., et al. (2008). Directed Evolution of a Lysosomal Enzyme with Enhanced Activity at Neutral pH by Mammalian Cell-Surface Display. Chemistry & Biology, 15(12), 1277-1286; b) {hacek over (S)}trojsová, A., & Dyhrman, S. T. (2008). Cell-specific β-N-acetylglucosaminidase activity in cultures and field populations of eukaryotic marine phytoplankton. FEMS Microbiology Ecology, 64(3), 351-361). The reason relates to the intrinsic hydrolysis instability of phenolic glycosides and in particular those constructed from electron-depleted phenols, such as dichloro-HPQ. It is effectively known that any nitrophenol-based glycoside (a phenol having the same electron depletion as dichloro-HPQ) hydrolyses spontaneously at physiological pH. It is also known that this stability problem is severely aggravated at more acid pH values (e.g. at a pH of 6.5), compared with physiological pH (pH 7.4).
Solutions leading to greater even complete absence of spontaneous degradation of the probes and hence of the production of erroneous signals, a fundamental prerequisite for use thereof for in vitro and in vivo applications, have been put forward involving the construction of a three-component probe. These comprise a para-hydroxy-or para-amino-benzyl spacer grafted on the phenolic fluorophore (WO2008145830). Although such para-hydroxybenzyl spacers have already been used in the development of prodrugs since the early 1980s (Wakselman, M. New Journal of Chemistry (1983), 7, 439-447), they have a recognized major drawback: the use of para-hydroxybenzyl spacers in artificial enzyme substrates leads to permanent alkylation of the protein, often in the vicinity of or inside the catalytic site. This negative property was subsequently put to advantage in numerous articles which proposed enzyme-inactivating substrates i.e. leading to an enzyme which is no longer capable of converting the substrate molecules (Zhu, J.; Withers, S. G.; Reichardt, P. B.; Treadwell, E.; Clausen, T. P. Biochem. J. 1998, 332, 367-371), as shown in following Scheme 1.

The different studies on enzymatic labelling by ortho- or para-hydroxybenzyl enzyme substrates show that quinone methides are highly reactive species which alkylate the nucleophiles and risk randomly modifying the molecular properties of the biomacromolecules lying in their immediate vicinity. The reduced activation or catalytic capabilities of the target enzyme are evidently most harmful for the sensitivity of imaging experiments using a said fluorogenic probe since enzymatic amplification is lost. Aside from the direct inactivation of the target enzyme, random alkylation of the surface of this protein or of any other neighbouring protein present also carries the risk of an immune response. Both cases generate limited tolerance by the respective body hence subjecting it to high toxicity (Grinda M. et al. ChemMedChem 2011, 6, 2137-2141)